Abstract Inositol 1,4,5-trisphosphate receptors (IP3Rs) are critical regulators of cell death in response to multiple stimuli, including engagement of the Fas death receptor (FasR). Our past work on this project discovered that the FasR directly engages and activates components of the T cell receptor complex to elicit apoptotic calcium release from IP3R channels. More recently, we demonstrated that very rapid Fas-dependent palmitoylation of the Src kinase Lck is essential for recruiting this protein into lipid rafts and initiating Fas-dependent calcium release and cell death. Surprisingly, Lck was depalmitoylated with equally rapid kinetics in the continued presence of Fas ligand. The kinetics of palmitoylation/depalmitoylation was temporally consistent with the phosphorylation/dephosphorylation of downstream signaling proteins such as phospholipase C gamma-1. Thus, we propose that palmitoylation is functionally analogous to phosphorylation and is responsible for the rapid recruitment and assembly of the FasR macromolecular signaling complex. The central hypothesis of this proposal is that dynamic and highly regulated lipidation of multiple signaling proteins is essential for signaling through the FasR. This hypothesis will be tested in three Specific Aims. Aim 1 will determine if rapid stimulus-dependent palmitoylation/depalmitoylation of components of the Fas signaling pathway contributes to apoptotic calcium release. We will exploit our recently developed and highly sensitive pulse labeling technique using alkyne lipids combined with acyl-biotin exchange to determine dynamic changes in the palmitoylated proteins after Fas stimulation. Aim 2 will determine how palmitoylation is enzymatically regulated after Fas stimulation. We will develop new methodological approaches to determine how the palmitoylating enzyme DHHC21and the expanding family of depalmitoylating enzymes are regulated during Fas stimulation. In Aim 3 we will determine if the DHHC21 mutant mouse depilated (dep) has deficits in T cell function in vitro and in vivo. We will evaluate T cell differentiation and function in dep mice and determine whether palmitoylation of FasR-associated proteins and downstream signaling are compromised in these mice. Together, the proposed studies will likely lead to new insights into the dynamic regulation of protein function by lipidation. Furthermore, these studies highlight an entirely new class of signaling proteins regulating T cell function which are potential therapeutic targets for diseases associated with altered T cell homeostasis such as autoimmunity and cancer.